חלק 1: הכנת הערוצים microfluidic צלחת BioFlux. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לפני הפעלת הניסוי התא זרימה, צריך להכין את ערוצי microfluidic עם ציפוי חלבון של עניין. במקרה זה, קולגן אני ישמשו. כל ערוץ יש מפרצון ולשקע היטב. עבור צלחת זו, כניסת גם הוא עזב גם להאכיל את ערוץ ולשקע גם הוא בצד ימין. </li><li style=";text-align:right;direction:rtl"> לדלל את הקולגן אני (5mg/ml המלאי) ציפוי ריכוז 200μg/ml של חומצה אצטית 0.02M. אחת תצטרך 20μl לכל ערוץ בשימוש. מערבבים על ידי triteration עדין עם טיפ micropipette. </li><li style=";text-align:right;direction:rtl"> הוסף 20 μl של ציפוי על כל ערוץ בהתאמה לשמש. צריך לוותר הנוזל לתוך להקיש הפנימי של משקע גם להאכיל את ערוץ microfluidic עניין באמצעות micropipette. הימנע מציגה בועות, לא לדחוף את האוויר מתוך פיפטה. כלול ערוץ אחד ללא קולגן שליטה לא קולגן (שליטה שלילי הידבקות וצבירה). </li><li style=";text-align:right;direction:rtl"> חבר את ממשק הצלחת על ידי האצבע הראשונה הידוק 4 ברגים ואז כל פעם הם קבוצה, השתמש הנהג מומנט לחלוטין להדק. הנהג מומנט ילחצו כאשר הוא מגיע אטימות מירבית. </li><li style=";text-align:right;direction:rtl"> שימוש במצב ידני בתוכנה BioFlux, להחיל זלוף לערוצים של ריבית 2dyn/cm 2-משקע היטב. כאן צריך להיזהר אגרוף הפנימי מול להתמלא בנוזל. זה ייקח כמה דקות. אפשר גם לראות את זה להתרחש באמצעות המטרה צריכת חשמל נמוכה על מיקרוסקופ (4X) ולמצוא את כניסת היטב או על ידי מחזיק את הצלחת להסתכל לתוך הבארות כניסת מלמטה. אחת צריך קודם לראות בטיפת נוזל לאט ממלא את האגרוף הפנימית. לאחר אגרוף הפנימי כניסת מלא, זלוף להפסיק בבת אחת על ידי לחיצה להפסיק בתוכנה. </li><li style=";text-align:right;direction:rtl"> דגירה את הצלחת בטמפרטורת החדר למשך שעה אחת. </li><li style=";text-align:right;direction:rtl"> הסר את הממשק ולהוסיף 1 מ&quot;ל של PBS (בתוספת Ca 2 + / Mg 2 +) לשקע היטב. התחל זלוף משקע היטב 2dyn/cm 2. המשך זלוף במשך 10 דקות. עצור זלוף ולהסיר את הממשק. </li><li style=";text-align:right;direction:rtl"> הסר PBS עודף כניסת והן בארות לשקע - אף פעם לא להסיר את נוזל הממלא את האגרוף הפנימית. חסום את הערוצים באמצעות פתרון חסימה (0.5% v / v BSA ב PBS (בתוספת Ca 2 + / Mg 2 +)). הוסף 1 מ&quot;ל של תמיסת חסימת לשקע כל טוב כדי לשמש perfuse משקע היטב 2dyn/cm 2. המשך זלוף במשך 10 דקות. עצור זלוף ולהסיר את הממשק. ערוצי מוכן עד שלב זה יכול לשמש לאורך כל היום. </li><li style=";text-align:right;direction:rtl"> לפני הוספת דם, להסיר את נוזל משני צדי הערוץ למעט אגרופים הפנימית. </li></ol> חלק 2. הכנת דם עם נוגדנים GPIIb / IIIa מעכבות. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בעוד הקולגן הוא דוגרים בערוצים, חייבים להכין את כל הדם. אחד צריך לעקוב אחר תקנות biosafety מוכתב על ידי המכון שלו / שלה בעת הטיפול דם אדם. </li><li style=";text-align:right;direction:rtl"> דם אדם טרי (מאדם בצום) להיגרר קרישה ציטרט הנתרן יש להשתמש בתוך 3 שעות של האוסף. </li><li style=";text-align:right;direction:rtl"> הכן דם על ידי הוספת calcein 4μM PM. הכינו מלאי של 4 מ&quot;מ calcein AM ב DMSO ולהוסיף דם דילול של 1 / 1000 v / נ מערבבים על ידי היפוך עדין. </li><li style=";text-align:right;direction:rtl"> לוותר על 1 מ&quot;ל של calcein AM שכותרתו דם לתוך 10 - 1.5mL microtubes. הוסף GPIIb / IIIa הנוגדן מעכב זה בדילול הרצוי (המשקל המולקולרי של IgG הוא ~ 150kDa). דוגמה לכך היא סדרת דילול מ 1200nM כדי 9nM וכולל בקרת אין נוגדנים ואת פקד הנוגדן קשור. לשלוט חיובי מצוין עיכוב יהיה ReoPro (abciximab, Eli Lilly) בריכוז של 20ug/ml. Mix ידי היפוך עדין. </li><li style=";text-align:right;direction:rtl"> דגירה בטמפרטורת החדר למשך שעה 1. מערבבים על ידי היפוך עדין כל 10 דקות. </li></ol> חלק 3: הפעלת התא זרימה ניסוי בתחנת העבודה BF1000. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ראשית, יש להגדיר את פרוטוקול אוטומטי במודול הבקרה BioFlux. עבור קולגן אני, יש להגדיר פרוטוקול לרוץ 10dyn/cm 2 במשך 10 דקות משקע היטב. בשלב זה, צריך גם להגדיר פרמטרים נתונים הרכישה באמצעות תחנת BF1000, כולל עמדות הערוץ, לתפוס גל FITC ואת זמן לשגות מידע. מומלץ לתפוס 3 בתחומי להציג באמצעות מטרה 10x לערוץ כל 30 שניות על פני משך זמן של 10 דקות בסך הכל. </li><li style=";text-align:right;direction:rtl"> עבודה במהירות, 500ul של דם מוכן של כל תנאי לאפיקים מוכן להפריד מקום. אין מקום דם מלאה נוגדנים לתוך ערוץ זה מצופה קולגן זו חסומה עתה. </li><li style=";text-align:right;direction:rtl"> מניחים את ממשק על הצלחת. </li><li style=";text-align:right;direction:rtl"> מניחים את הצלחת על המיקרוסקופ עבודה BF1000. קלאמפ על הממשק. </li><li style=";text-align:right;direction:rtl"> בצע התאמה עומס הצלחת. כמו כן, מהלך הבמה באר אחת המכילה דם. הגדר FTIC ללכוד תמונה הפרמטרים (זמן חשיפה וכו &#39;לזכות ...). </li><li style=";text-align:right;direction:rtl"> בתוכנה BioFlux מונטאז&#39;, להתחיל רכישת להתחיל לזרום ורכישת בו זמנית. </li><li style=";text-align:right;direction:rtl"> הסר את צלחת מתחנת עבודה BF1000, להיפטר צלחת בהתאם להנחיות מוסדיים. </li></ol> חלק 4: תוצאות נציג. הנה פרוטוקול של הידבקות אגרגציה של טסיות דם בערוצים microfluidic הוצג. טיפול מעכב אגרגציה של טסיות דם, anti-GPIIb/IIIa נכלל גם בפרוטוקול. באמצעות ערוצי קולגן מצופה microfluidic של מערכת BioFlux, יש לצפות היווצרות פקיק אגרסיבי לאורך זמן עם דגימת דם לא מטופל מלאה ללא היווצרות פקיק בערוץ ללא ציפוי. באחד הניסויים שבוצעו לאחרונה, הגודל הממוצע של אגרגטים בתנאים השליטה 2000μm 2. באיור 1. <img src="/files/ftp_upload/1644/1644fig1.jpg" alt="איור 1" /> הופעל GPIIb / IIIa הוא מתווך חזק של טסיות-טסיות אינטראקציות צבירה ייצוב 1. הדבקה על קולגן מפעיל את מורכבות GPIIb / IIIa לעורר את התגובה הזאת 2. לאחר הדגרה עם anti-GPIIb/IIIa שעה 1 לפני החשיפה גזירה, יש לצפות לצפות לירידה בגודל של thrombi כמו גם ירידה בשכיחות של היווצרות פקיק. התגובה תלויה במינון ניתן לבצע בדרך כלל שנצפה 10dyn/cm 2. איור 2. <img src="/files/ftp_upload/1644/1644fig2.jpg" alt="איור 2" /> הערך 50 IC עבור מעכב המסוים הזה היה 17nM ב 10 dyn / 2 ס&quot;מ. עיכוב מרבית (עבור התורם הזה) לעומת שליטה אין את הנוגדן היה 11% ב 10 dyn / 2 ס&quot;מ. בעוד שיטות כאן הוצגו באמצעות חלבון מסוים תאי מטריקס ו מעכב ספציפי של אגרגציה של טסיות דם, הפרוטוקול הוא להרחבה ציפויים אחרים, סוגי תאים אחרים מעכבי אחרים עבור מבחני הידבקות התא. המרכיבים המפתח להצלחה של ניסויים כאלה הימנעות בועות ערוצי microfluidic, דילול נכון של כל ריאגנטים ב diluents נכונה, תנאי הדגירה מתאים ריאגנטים בכלל.

<ol>
	<li>Jackson, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+growing+complexity+of+platelet+aggregation.">The growing complexity of platelet aggregation.</a> Blood. 109, 5087-5095 (2007).</li><li>Nakamura, T., Kambayashi, J., Okuma, M., Tandon, N. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+the+GP+IIb-IIIa+complex+induced+by+platelet+adhesion+to+collagen+is+mediated+by+both+alpha2beta1+integrin+and+GP.">Activation of the GP IIb-IIIa complex induced by platelet adhesion to collagen is mediated by both alpha2beta1 integrin and GP.</a> VI. J Biol Chem. 274, 11897-11903 (1999).</li></ol>

הכותבים הם עובדי תורם של Fluxion Biosciences.

בעוד שיטות כאן הוצגו באמצעות חלבון מסוים תאי מטריקס ו מעכב ספציפי של אגרגציה של טסיות דם, הפרוטוקול הוא להרחבה ציפויים אחרים, סוגי תאים אחרים מעכבי אחרים עבור מבחני הידבקות התא. המרכיבים המפתח להצלחה של ניסויים כאלה הימנעות בועות ערוצי microfluidic, דילול נכון של כל ריאגנטים ב diluents נכונה, תנאי הדגירה מתאים ריאגנטים בכלל.

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>collagen I, Bovine</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>A1064401</td>
<td>other sources of collagen I can be used</td>
</tr>
<tr>
<td>PBS plus Ca/Mg</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>14040-117</td>
<td>&nbsp;</td>
<tr>
<td>Bovine serum albumin (BSA)</td>
<td>Reagent</td>
<td>EMD</td>
<td>EM-2930 Bottom of Form</td>
<td>From VWR</td>
</tr>
<tr>
<td>calcein AM</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>C1430</td>
<td>Calcein AM from BD can also be used</td>
</tr>
<tr>
<td>BioFlux 1000 System</td>
<td>Reagent</td>
<td>Fluxion Biosciences</td>
<td>Contact Company</td>
<td>&nbsp;</td>
<tr>
<td>BioFlux Plate</td>
<td>Reagent</td>
<td>Fluxion Biosciences</td>
<td>900013</td>
<td>&nbsp;</td>
<tr>
<td>antiGPIIb/IIIa</td>
<td>Reagent</td>
<td>Abcam</td>
<td>ab33407</td>
<td>an alternative # is ab15021</td>
</tr>
<tr>
<td>ReoPro</td>
<td>Reagent</td>
<td>Eli Lilly</td>
<td>&nbsp;</td>
<td>by Rx only</td>
</tr>		</tbody>
		</table>
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platelet adhesion and aggregation under flow using microfluidic flow cells

צבירת טסיות דם מתרחשת בתגובה לפגיעה בכלי הדם שבו תאי מטריקס להלן האנדותל נחשף. מפל הידבקות טסיות מתקיים בנוכחות זרימה גזירה, גורם לא היוו ב קונבנציונאלי (סטטי) היטב הצלחת מבחני. מאמר זה מדווח על assay טסיות צבירה תוך שימוש בתבנית microfluidic צלחת היטב לחקות פיסיולוגי תנאי הזרימה גזירה. חלבונים תאיים, קולגן אני או גורם פון Willebrand מופקדים בתוך הערוץ microfluidic באמצעות זלוף פעיל עם משאבה פנאומטי. חלבונים מטריצה ​​נשטפים אז עם חיץ וחסמו להכין את הערוץ microfluidic עבור אינטראקציות הטסיות. דם שלם שכותרתו עם צבע פלואורסצנטי הוא perfused דרך ערוץ ברמות ספיקה שונים על מנת להשיג ההפעלה אגרגציה של טסיות דם. צבירה של מעכבי טסיות ניתן להוסיף לפני הניסוי תזרים התא לייצר IC50 בתגובה לנתונים מנה.

Watch this Scientific Journal Video about Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells at JoVE.com

Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells

מפל הידבקות טסיות מתקיים בנוכחות זרימה גזירה, גורם לא היוו ב קונבנציונאלי (סטטי) היטב הצלחת מבחני. מאמר זה מדווח על assay טסיות צבירה תוך שימוש בתבנית microfluidic צלחת היטב לחקות פיסיולוגי תנאי הזרימה גזירה.

הדבקה צבירת טסיות דם תחת זרימה באמצעות תאים תזרים Microfluidic

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion ...

platelet-adhesion-aggregation-under-flow-using-microfluidic-flow

Universidad San Sebastian

Research

JoVE Journal

Biology

18.1K Views.  Fluxion Biosciences, Inc.. מפל הידבקות טסיות מתקיים בנוכחות זרימה גזירה, גורם לא היוו ב קונבנציונאלי (סטטי) היטב הצלחת מבחני. מאמר זה מדווח על assay טסיות צבירה תוך שימוש בתבנית microfluidic צלחת היטב לחקות פיסיולוגי תנאי הזרימה גזירה.

Video: Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

Cytometry זרימה טיהור תאים עכבר meiotic

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

אופטימיזציה שיטות דוגמנות מכתימה והפצת נשק לניטור חלוקת תא באמצעות צבעי מעקב סלולרי

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

4D Imaging of Protein Aggregation in Live Cells

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental conditions and an intracellular environment that is tightly packed, sticky, and hazardous to protein stability1. The ability to dynamically balance protein production, folding and degradation demands highly-specialized quality control machinery, whose absolute necessity is observed best when it malfunctions. Diseases such as ALS, Alzheimer's, Parkinson's, and certain forms of Cystic Fibrosis have a direct link to protein folding quality control components2, and therefore future therapeutic development requires a basic understanding of underlying processes. Our experimental challenge is to understand how cells integrate damage signals and mount responses that are tailored to diverse circumstances.
The primary reason why protein misfolding represents an existential threat to the cell is the propensity of incorrectly folded proteins to aggregate, thus causing a global perturbation of the crowded and delicate intracellular folding environment1. The folding health, or "proteostasis," of the cellular proteome is maintained, even under the duress of aging, stress and oxidative damage, by the coordinated action of different mechanistic units in an elaborate quality control system3,4. A specialized machinery of molecular chaperones can bind non-native polypeptides and promote their folding into the native state1, target them for degradation by the ubiquitin-proteasome system5, or direct them to protective aggregation inclusions6-9.
In eukaryotes, the cytosolic aggregation quality control load is partitioned between two compartments8-10: the juxtanuclear quality control compartment (JUNQ) and the insoluble protein deposit (IPOD) (Figure 1 - model). Proteins that are ubiquitinated by the protein folding quality control machinery are delivered to the JUNQ, where they are processed for degradation by the proteasome. Misfolded proteins that are not ubiquitinated are diverted to the IPOD, where they are actively aggregated in a protective compartment.
Up until this point, the methodological paradigm of live-cell fluorescence microscopy has largely been to label proteins and track their locations in the cell at specific time-points and usually in two dimensions. As new technologies have begun to grant experimenters unprecedented access to the submicron scale in living cells, the dynamic architecture of the cytosol has come into view as a challenging new frontier for experimental characterization. We present a method for rapidly monitoring the 3D spatial distributions of multiple fluorescently labeled proteins in the yeast cytosol over time. 3D timelapse (4D imaging) is not merely a technical challenge; rather, it also facilitates a dramatic shift in the conceptual framework used to analyze cellular structure.
We utilize a cytosolic folding sensor protein in live yeast to visualize distinct fates for misfolded proteins in cellular aggregation quality control, using rapid 4D fluorescent imaging. The temperature sensitive mutant of the Ubc9 protein10-12 (Ubc9ts) is extremely effective both as a sensor of cellular proteostasis, and a physiological model for tracking aggregation quality control. As with most ts proteins, Ubc9ts is fully folded and functional at permissive temperatures due to active cellular chaperones. Above 30 &deg;C, or when the cell faces misfolding stress, Ubc9ts misfolds and follows the fate of a native globular protein that has been misfolded due to mutation, heat denaturation, or oxidative damage. By fusing it to GFP or other fluorophores, it can be tracked in 3D as it forms Stress Foci, or is directed to JUNQ or IPOD.

Cellular viability depends on timely and efficient management of protein misfolding. Here we describe a method for visualizing the different potential fates of a misfolded protein: refolding, degradation, or sequestration in inclusions. We demonstrate the use of a folding sensor, Ubc9ts, for monitoring proteostasis and aggregation quality control in live cells using 4D microscopy.

4D הדמיה של צבירת חלבון בתאים חיים

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental ...

Reduced-gravity Environment Hardware Demonstrations of a Prototype Miniaturized Flow Cytometer and Companion Microfluidic Mixing Technology

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If humans are to travel beyond low Earth orbit, a transition towards space-ready, point-of-care (POC) testing is required. Such testing needs to be comprehensive, easy to perform in a reduced-gravity environment, and unaffected by the stresses of launch and spaceflight. Countless POC devices have been developed to mimic laboratory scale counterparts, but most have narrow applications and few have demonstrable use in an in-flight, reduced-gravity environment. In fact, demonstrations of biomedical diagnostics in reduced gravity are limited altogether, making component choice and certain logistical challenges difficult to approach when seeking to test new technology. To help fill the void, we are presenting a modular method for the construction and operation of a prototype blood diagnostic device and its associated parabolic flight test rig that meet the standards for flight-testing onboard a parabolic flight, reduced-gravity aircraft. The method first focuses on rig assembly for in-flight, reduced-gravity testing of a flow cytometer and a companion microfluidic mixing chip. Components are adaptable to other designs and some custom components, such as a microvolume sample loader and the micromixer may be of particular interest. The method then shifts focus to flight preparation, by offering guidelines and suggestions to prepare for a successful flight test with regard to user training, development of a standard operating procedure (SOP), and other issues. Finally, in-flight experimental procedures specific to our demonstrations are described.

Spaceflight blood diagnostics need innovation. Few demonstrations have been published illustrating in-flight, reduced-gravity health diagnostic technology. Here we present a method for construction and operation of a parabolic flight test rig for a prototype point-of-care flow-cytometry design, with components and preparation strategies adaptable to other setups.

הפגנות חומרת הסביבה מופחתת כובד של טכנולוגיה cytometer זרימה וCompanion Microfluidic אב טיפוס מוקטנת ערבוב

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If ...

LED Thermo Flow &#8212; Combining Optogenetics with Flow Cytometry

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the controlled delivery of light to the cell sample and hence the most popular tools for optogenetic studies are microscopy-based cell analyses and in vitro experiments. The flow cytometer has major advantages over a microscope, including the ability to rapidly measure thousands of cells at single cell resolution. However, it is not yet widely used in optogenetics. Here, we present a device that combines the power of optogenetics and flow cytometry: the LED Thermo Flow. This device illuminates cells at specific wavelengths, light intensities and temperatures during flow cytometric measurements. It can be built at low cost and be used with most common flow cytometers. To demonstrate its utility, we characterized the photoswitching kinetics of Dronpa proteins in vivo and in real time. This protocol can be adapted to almost all optically controlled substances and substantially expands the set of possible experiments. More importantly, it will greatly simplify the discovery and development of new optogenetic tools.

LED Thermo Flow &amp;#8212; Combining Optogenetics with Flow Cytometry

Optically controlled substances are powerful tools to study signaling pathways. To expand the spectrum of possible experiments, we developed a device for studying optically controlled substances in real time using flow cytometry: the LED Thermo Flow.

LED Thermo Flow - שילוב Optogenetics עם cytometry זרימה

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

ניתוח של המתלים תא מבודד מרקמות Solid ידי זרימת cytometry ספקטרלי

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Live-cell Imaging of Platelet Degranulation and Secretion Under Flow

Blood platelets are essential players in hemostasis, the formation of thrombi to seal vascular breaches. They are also involved in thrombosis, the formation of thrombi that occlude the vasculature and injure organs, with life-threatening consequences. This motivates scientific research on platelet function and the development of methods to track cell-biological processes as they occur under flow conditions.
A variety of flow models are available for the study of platelet adhesion and aggregation, two key phenomena in platelet biology. This work describes a method to study real-time platelet degranulation under flow during activation. The method makes use of a flow chamber coupled to a syringe-pump setup that is placed under a wide-field, inverted, LED-based fluorescence microscope. The setup described here allows for the simultaneous excitation of multiple fluorophores that are delivered by fluorescently labeled antibodies or fluorescent dyes. After live-cell imaging experiments, the cover glasses can be further processed and analyzed using static microscopy (i.e., confocal microscopy or scanning electron microscopy).

This work describes a fluorescence microscopy-based method for the study of platelet adhesion, spreading, and secretion under flow. This versatile platform enables the investigation of platelet function for mechanistic research on thrombosis and hemostasis.

Live-cell הדמיה של degranulation טסיות והפרשה תחת זרימה

Blood platelets are essential players in hemostasis, the formation of thrombi to seal vascular breaches. They are also involved in thrombosis, the ...

Quantification of Cellular Densities and Antigenic Properties using Magnetic Levitation

The described method was developed based on the principles of magnetic levitation, which separates cells and particles based on their density and magnetic properties. Density is a cell type identifying property, directly related to its metabolic rate, differentiation, and activation status. Magnetic levitation allows a one-step approach to successfully separate, image and characterize circulating blood cells, and to detect anemia, sickle cell disease, and circulating tumor cells based on density and magnetic properties. This approach is also amenable to detecting soluble antigens present in a solution by using sets of low- and high-density beads coated with capture and detection antibodies, respectively. If the antigen is present in solution, it will bridge the two sets&#160;of beads, generating a new bead-bead complex, which will levitate in between the rows of antibody-coated beads. Increased concentration of the target antigen in solution will generate a larger number of bead-bead complexes when compared to lower concentrations of antigen, thus allowing for quantitative measurements of the target antigen. Magnetic levitation is advantageous to other methods due to its decreased sample preparation time and lack of dependance on classical readout methods. The image generated is easily captured and analyzed using a standard microscope or mobile device, such as a smartphone or a tablet.

This paper describes a magnetic levitation-based method that can specifically detect the presence of antigens, either soluble or membrane-bound, by quantifying changes in the levitation height of capture beads with fixed densities.

כימות של צפיפות תאית ומאפיינים אנטיגניים באמצעות ריחוף מגנטי

The described method was developed based on the principles of magnetic levitation, which separates cells and particles based on their density and magnetic ...

Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from Hematopoietic Progenitors

Megakaryocyte (MK) differentiation encompasses a number of endomitotic cycles that result in a highly polyploid (reaching even&#160;&#62;64N) and extremely large cell (40-60 &#181;m). As opposed to the fast-increasing knowledge in megakaryopoiesis at the cell biology and molecular level, the characterization of megakaryopoiesis by flow cytometry is limited to the identification of mature MKs using lineage-specific surface markers, while earlier MK differentiation stages remain unexplored. Here, we present an immunophenotyping strategy that allows the identification of successive MK differentiation stages, with increasing ploidy status, in human primary sources or in vitro cultures with a panel integrating MK specific and non-specific surface markers. Despite its size and fragility, MKs can be immunophenotyped using the above-mentioned panel and enriched by fluorescence-activated cell sorting under specific conditions of pressure and nozzle diameter. This approach facilitates multi-Omics studies, with the aim to better understand the complexity of megakaryopoiesis and platelet production in humans. A better characterization of megakaryopoiesis may pose fundamental in the diagnosis or prognosis of lineage-related pathologies and malignancy.

Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from Hematopoietic Progenitors

Here, we present an immunophenotyping strategy for the characterization of megakaryocyte differentiation, and show how that strategy allows the sorting of megakaryocytes at different stages with a fluorescence-activated cell sorter. The methodology can be applied to human primary tissues, but also to megakaryocytes generated in culture in vitro.

אימונופנוטיפינג ומיון תאים של חברי כנסת אנושיים ממקורות ראשוניים אנושיים או מובחנים במבחנה מאבות ההמטופיאטיים

Megakaryocyte (MK) differentiation encompasses a number of endomitotic cycles that result in a highly polyploid (reaching even&#160;&#62;64N) and ...

Isolation and Identification of Vascular Endothelial Cells from Distinct Adipose Depots for Downstream Applications

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone regulation, barrier functions, and angiogenesis. Endothelial cell dysfunction is a hallmark predictor and major driver for the progression of severe cardiovascular diseases, yet the underlying mechanisms remain poorly understood. The ability to isolate and perform analyses on endothelial cells from various vascular beds in their native form will give insight into the processes of cardiovascular disease. This protocol presents the procedure for the dissection of mouse subcutaneous and mesenteric adipose tissues, followed by isolation of their respective arterial vasculature. The isolated arteries are then digested using a specific cocktail of digestive enzymes focused on liberating functionally viable endothelial cells. The digested tissue is assessed by flow cytometry analysis using CD31+/CD45&#8722; cells as markers for positive endothelial cell identification. Cells can be sorted for immediate downstream functional assays or used to generate primary cell lines. The technique of isolating and digesting arteries from different vascular beds will provide options for researchers to evaluate freshly isolated vascular cells&#160;from arteries of interest and allow them to perform a wide range of functional tests on specific cell types.

This protocol details a method for the dissection of mouse adipose depots and the isolation and digestion of respective arteries to liberate and then identify the endothelial cell population. Freshly isolated cells used in downstream applications will advance the understanding of vascular cell biology and the mechanisms of vascular dysfunction.

בידוד וזיהוי של תאי אנדותל וסקולריים ממחסני שומן נפרדים ליישומים במורד הזרם

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone ...